The invention relates to a preparative electrophoresis device and methods of using it.
Electrophoresis involves the use of an electric field to cause different charged molecules in a sample to migrate through a gel at different rates, resulting in separation of the different molecules. Separation is based on charge and/or size differences between different molecules. Molecules with higher charge migrate faster than molecules with lower charge, and lower molecular weight molecules migrate faster than the higher molecular weight molecules.
Because of the high resolving power of electrophoresis, it is widely used to separate proteins or peptides and polynucleotides, both as an analytical technique (in which molecules remain in the gel) and as a preparative tool (in which molecules are recovered). The gel can be cylindrical in a tubular chamber, or it can be flat in the form of a slab between two plates. Some commercially available analytical slab devices use disposable prepoured gels with thicknesses ranging between 0.5 and 1.5 mm, and plates generally about 8 cm high and about 10 cm wide; these are generally known as "mini-gel" systems. Some larger analytical gels employ spacers 1.5 mm thick with plates generally about 15 cm high and 18 cm wide.
The molecules separated by electrophoresis can be recovered from the gel by slicing the gel or by electrophoretic transfer onto a membrane support. Further processing is usually required to remove the molecules from the gel or membrane support for further purification procedures. This preparative procedure is known as zone excision extraction.
The separated molecules can also be recovered by having them pass from the gel into a collection channel and flushing the channel with a buffer. Because different molecules enter the channel at different times they will be eluted separately. This preparative procedure is known as successive zone elution and requires the use of a special gel construction to provide a collection channel. These systems are typically of the tube gel type, and often employ a cooling system to dissipate the heat that is generated and can otherwise affect electrophoresis owing to increased temperature. The Model 491 system available from Bio-Rad Laboratories is an example of such a system. Chen U.S. Pat. No. 4,877,510 describes a cylindrical electrophoretic column that has a chamber defined by a porous plate and a semi-permeable membrane thereunder for receiving and removing molecules from the bottom of the column; electrophoretic buffer passes through the membrane into the porous plate and carries the molecules with it when it is pumped out.
Slab systems have also been used for successive zone elution preparative electrophoresis. Carpenter, H. P. et al., "A Device for Preparative Elution Electrophoresis Using a Polyacrylamide Gel Slab", Electrophoresis, Vol. 7, pp. 221-226 (1986) describes a device that is clipped on to the end of a 3 mm thick polyacrylamide slab gel and employs two paper membranes impregnated with polyacrylamide and separated by a 0.5 mm gap. The top membrane has 4% polyacrylamide, and the lower one has 25% polyacrylamide, permitting proteins of M.sub.r 10,000 to 1,000,000, to pass through the top membrane to the space between them, but to not pass through the lower membrane. Electrode buffer passes through the chamber to remove molecules migrating from the bottom of the gel into the compartment. This device is also described in UK Published Patent Application No. 2177211A and U.S. Pat. No. 4,707,233. Groschup, M. H., et al., "A Convenient Gel Holder for Preparative Electrophoretic Separation of Aggregated Bacterial Proteins", Electrophoresis. Vol. 12, pp. 90-91 (1991) describes a slab electrophoresis device having a transverse horizontal channel formed in the middle of the gel to collect proteins; inlet and outlet tubes are connected to the channel via threaded fittings.